1. Field of the Invention
This invention relates to stabilized probiotic compositions, including synbiotic compositions and compositions used in conjunction with acidified drinking water, and a system and method for delivering the compositions to animals and plants.
2. Description of Related Art
Probiotics have been used in farm and agricultural applications for many years. A primary use for probiotic formulations is as a feed additive, but other uses include the treatment of housing, animal wound care, pond treatments, and water treatment. When used as a feed additive, the probiotic material is typically added directly to the feed (known as direct-fed microbials or “DFM”) and consumed by the animal. DFM products are commercially available in a variety of product forms, including powder, paste, gel, bolus, and capsules, which may be mixed in feed, top-dressed, given as a paste, or mixed into drinking water or a milk replacer. Usage doses vary by product, from single dose applications to continuous feeding application. Most DFM products must be stored in a cool, dry area away from heat, direct sunlight, and high levels of humidity to avoid damaging the bacteria or rendering the bacteria ineffective as a probiotic. Some forms of commercially available DFM products contain bacterial spores, particularly Bacillus species, which are considered more stable and may have long shelf lives even under harsh environmental conditions, such as elevated temperatures, dryness and pH extremes. These spores will germinate into vegetative cells and grow when conditions become favorable. Several Bacillus species are approved by the Food & Drug Administration and the Association of American Feed Control Officials (AAFCO) for use in DFM products in the U.S., including B. subtilis, B. licheniformis, B. pumilus, B. coagulans, and B. lentus. Other countries have approved use of other Bacillus species as probiotic microorganisms.
Even relatively stable bacterial species used in DFM products may be sensitive to certain conditions typically found in application of DFM products to livestock. For example, the water activity in the feed can reduce the shelf-life of even the most stable bacterial forms. Various techniques are known to help increase shelf life of the bacteria in DFM products. Microencapsulation is one known method to increase shelf life of probiotic formulas to allow use in DFM products. For example, U.S. Pat. No. 6,254,910 discloses a delivery system for delivering unstable or sensitive ingredients, wherein the unstable or sensitive ingredient is coextruded with other food ingredients so that it is encapsulated within an outer layer of food ingredients, with each layer having specific moisture contents. Several probiotic species, including Bacillus coagulans, preferably in spore form, are disclosed in the '910 patent as unstable or sensitive ingredients that are suitable for encapsulation to protect the probiotic and increase the probability of survival during processing and in the environmental conditions of use. Other encapsulation technologies including spray drying extrusion, emulsion and phase separation have been used, but with limited success and added expense. A known encapsulated probiotic product, known as Bac-In-A-Box, is commercially available from SG Austria (http://www.sqaustria.com/probiotics). Newer microencapsulation techniques using calcium-alginate gel capsule formation appear promising, but are still in the development stage and are not yet suitable for large industrial applications.
Similarly, U.S. Pat. No. 5,501,857 discloses a capsule within a capsule having bacterial species, such as one from the genera Lactobacillus or Bifidobacterium, in the inner capsule and other beneficial ingredients, such as vitamins, in the outer capsule, with each capsule being surrounded by a gelatin shell. The capsule within a capsule structure allows the use of multiple beneficial ingredients that are not compatible within a single capsule. These capsules have the drawback of requiring direct oral administration to the animals. Similar direct oral administration compositions and methods are disclosed in WO 2010/079104, WO 2012/167882, and U.S. Patent Application Publication No. 2013/0017174. However, these formulas are generally complex and must be administered directly to each animal, which is time consuming and may be difficult.
It is also known to add probiotic bacteria to animal drinking water using a biogenerator to produce the bacteria in a vegetative state. Such a method is disclosed in U.S. Pat. No. 4,910,024, which describes a biogenerator technology and method for delivering temperature-sensitive probiotic bacteria in a live, vegetative condition into a potentially large number of domestic animals as a means of increasing nutrient absorption efficiency and controlling the proliferation of harmful microorganisms in the digestive tracts of such animals. There are several drawbacks to the use of an on-site biogenerator to deliver probiotics. A primary drawback is the difficulty with maintaining sterility or preventing contamination from external sources of bacteria, yeast, and fungi. Ideally, the water used in the biogenerator must be pathogen free and the device must prevent airborne transmission of undesirable microorganisms, which may grow in the device and be administered to the animals. These devices also require power and sufficient water pressure. Additionally, the growth of bacteria in the biogenerator is temperature dependent and the device would need to be temperature controlled to ensure proper growth of the bacteria. The normal ambient temperature and environmental variations (such as humidity) at sites where the device would typically be used, such as a barn, would result in adverse growth variations and inconsistencies without temperature and environmental control. DFM products that deliver vegetative state bacteria to the animals' feed or drinking water might also be more susceptible to the harsh stomach environment once ingested by the animals and may not survive to reach the intestinal tract where the probiotics are generally most effective. Additionally, the quantity of bacteria must be fed to the animals at the appropriate time and in a proper concentration to be effective, and this can be difficult to achieve with existing probiotic delivery technology.
There are also known systems for delivery of sterile liquids. For example, U.S. Pat. No. 5,320,256 discloses a system for delivering a sterile liquid comprising a compressible reservoir for storing the sterile liquid, a flexible delivery element extending from the reservoir with the delivery element having a hollow interior, and a series of shut off valves to allow delivery and prevent backflow of the liquid or air into the system. The '256 patent is not specifically related to delivery of probiotic formulas or delivery of sterile liquids to animals or in agricultural settings. Another example, which is specifically for delivery of medicines, vitamins, nutrients, and the like to animals, is found in U.S. Pat. No. 6,723,076. The delivery system disclosed in the '076 patent comprises a sealed, collapsible bag with two flexible tubes attached for filling the bag and administering the solution from the bag to an animal with a syringe gun. Although these systems address some of the problems associated with probiotic delivery, they do not address problems related to the environmental stability of the probiotic composition or automated and controlled dosing of the probiotic composition to a feed or water supply.
It is also known to administer probiotics to animals in spore form. In addition to the '910 patent discussed above, U.S. Pat. No. 4,999,193 discloses adding spores of bacteria, particularly Bacillus cereus (IP 5832 strain), to animal feed or drinking water. Spores are known to be able to withstand high temperatures, making them better suited for incorporation into animal feed during manufacture of the feed, which typically involves heat. Although the use of spore form bacteria addresses the problems associated with temperature stability, the prior art does not provide an adequate delivery system that eliminates outside contamination while achieving controlled delivery in a manner that is easily used in existing facilities without requiring any retrofitting or additional power sources.
In addition to providing probiotics, it also known to provide prebiotics in combination with probiotics. This combination is generally known as synbiotics. For example, WO 2012/027214 discloses a synbiotic combination of spore form Bacillus bacteria with prebiotic carbohydrates, including arabinoxylan, arabinoxylan oligosaccharides, xylose, soluble fiber dextrin, soluble corn fiber, and polydextrose. A prebiotic is a non-digestible carbohydrate or soluble fiber that provides a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of gut beneficial bacteria and/or reducing pathogenic populations. Prebiotics are resistant to digestive gastric acid and digestive enzymes in the animal's stomach and small intestine and are able to reach the large intestine substantially intact or only partially degraded (other than dissolving in water present in the gastrointestinal tract). Once in the large intestine, the prebiotics provide a carbohydrate food source for beneficial bacteria and undergo complete or partial fermentation in the colon (part of the large intestine within which additional nutrient absorption occurs through the process of fermentation). Fermentation occurs by the action of bacteria within the colon on the prebiotic food mass, producing gases and short-chain fatty acids (SCFA). The production of SCFAs, such as butyric acid, acetic acid, propionic acid, and valeric acids, is increased when prebiotics are added to animal feed. Scientific studies have indicated that SCFAs have significant health benefits. By increasing beneficial bacterial populations, prebiotics also suppress the populations of pathogenic bacteria in the colon, such as Clostridia, E. Coli, and Salmonella. 
A variety of prebiotics are known, including polysaccharides, oligosaccharides, fructooligosaccharides (FOS), galactooligosaccharides (GOS), soya-oligosaccharides (SOS), xylo-oligosaccharides (XOS), pyrodextrins, isomalto-oligosaccharides (IMO), and lactulose. Specific water-soluble dietary fiber prebiotics also include Fructans (inulin), Xanthan Gum (E415), Pectin (E440), Natriumalginat (E401), Kaliumalginat (E402), Ammoniumalginat (E403), Calciumalginat (E404), PGA (E405), Agar (E406), and Carrageen (E407). Ingestion of these prebiotic fibers can change how other nutrients and chemicals are absorbed through bulking and viscositys and can also change the nature of the contents of the gastrointestinal tract, having been shown to increase populations of Lactobacilli and Bifidobacteria in the intestine and cecum of livestock. In poultry studies (particularly studies regarding broilers), providing a synbiotic combination of probiotic and prebiotic has been shown to increase the villus/crypt ratio (or ratio of villus height:crypt depth). The villus/crypt ratio is an indicator of the likely digestive capacity of the small intestine. It is within the small intestine that the final stages of enzymatic digestion occur, liberating small molecules capable of being absorbed, such as, sugars, monosaccharides, disaccharides, amino acids, dipeptides, and lipids. All of this absorption and much of the enzymatic digestion takes place on the surface of small intestinal epithelial cells, and to accommodate these processes, a huge mucosal surface area is required. The villi are minute (finger-like, hair-like, worm-like) projections from epithelial lining of the small intestine. Villus height is measured from the tip (top) of the villus to the villus-crypt junction. The villi are filled with blood vessels where the circulating blood takes picks up the nutrients. The crypt is the area between villi. Crypt depth is defined as the depth of the invagination, or in folding of the wall, between adjacent villi. Measurements for crypt depth are measured from the base upwards to the region of transition between the crypt and villus. Villus surface area is calculated by using formula, VW/2 times VL, where VW equals the villous width and VL equals villus length. More surface area provides more absorption of nutrients. The increase in villus/crypt ratio found in the poultry symbiotic study indicates an increase in digestion and absorption of nutrients.
Some studies have also shown the importance and benefits of this kind of synergy between probiotics and prebiotics and the effectiveness in helping young animals to achieve better growth performance. Studies using B. subtilis as the probiotic and inulin as the prebiotic have shown that the combination is more effective in swine and poultry populations than the use of B. subtilis alone. For example, this synbiotic combination was shown to reduce (P<0.05) excreta pH, intestinal digesta and cecal content pH compared with a control group. The combination also modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of Bifidobacteria and Lactobacilli compared with a control group. In another study, weaned piglets were shown to have increased levels of butyrate (a SOFA) when fed with a diet containing prebiotics. The importance of butyrate on gut improvement is well known, as this is crucial to optimize nutrient absorption.
Another issue encountered in animal and plant watering systems is bacteria populations in the drinking water and water transport systems. Municipal water supplies typically have some levels of bacteria present and local sources of water (such as an on-site pond) may contain bacteria from surrounding soil, fish, and run-off. These bacteria are known to result in or contribute to the formation of biofilms in the drinking water system. Biofilms are formed when microbial cells attach to surfaces in the water system, such as pipes and drinking nipples/nozzles, and form a film or slime layer. These biofilms can build-up, resulting in clogging parts of the system, or portions of the biofilm may also break-off causing additional clogging in other areas of the system, which reduce the amount of water available to the animals or plants. One known method for removing biofilms in these water systems is to flush the film by increasing the pressure in the water line. This method may cause damage to parts of the water system and typically leaves behind a mineral deposit from the biofilm, which will serve as a shelter for micro-organisms and result in the biofilm being reestablished. Chemical treatment products, such as chlorine and hydrogen peroxide are also known to be used and have good sanitizing abilities; however, these products are not beneficial to gut health for the animals or soil health for plants and may even be harmful.
It is also know to acidify the water by adding certain organic acids to the water. The use of acidified water is beneficial for several reasons, including that the acid, in its non-dissociated form, can penetrate through the bacterial wall and destroy certain microorganisms, which can reduce biofilm formation in the water system, aid in keeping drinking trough, nipples/nozzles clean, and can reduce the number of bacteria in the water. Since the bacteria in the water may be pathogenic and may cause illness when ingested by the animal, reduction of the bacteria in the water may be particularly helpful in light of bans on antibiotic use in certain geographic areas, such as Europe. Additionally, when ingested by animals in sufficient quantities to result in a stomach pH below about 6, the growth of pathogenic microorganisms (from other sources, such as food) is inhibited. Typically, weak organic acids, such as acetic acid, butyric acid, lactic acid, and sorbic acid, are used to acidify drinking water. There are a number of drawbacks to or difficulties with acidifying drinking water. For example, it can be difficult to maintain the pH level at a desired range (below 7 and usually below 5.5). Applying single acids in drinking water typically results in the pH decreasing quickly, which can have negative results, such as less water intake and decreased performance, including lower feed conversion rate and lower daily weight gain in the animals. For example, during a period of disease, pigs will drop their feed intake but maintain their water intake. Thus, palatable water is important for the GIT health of the animals. The use of acids can also be corrosive to metal components in the water system, resulting in added repair and replacement costs. To decrease these effects, a mix of organic acids may be used to acidify the drinking water, since the mix has a buffering effect that makes the pH decrease slowly. A synergistic mix of organic acids has also a greater antibacterial effect, is more tasteful, and is less corrosive when compared with single acids. Incorrect use of acidified drinking water can also result in proliferation of bacterial populations and growth of algae (which can result in further clogging of the water system) and reduction of feed intake (which can result in decreased weight gain and inadequate absorption of nutrients). Additionally, some acids are known to cause fungal growth, which can clog system parts and be detrimental to the animal.
Although it is known to use probiotics alone or in combination with prebiotics and to separately use acidified drinking water to provide health benefits to animals and to improve cleanliness in plant and animal water systems, it has not previously been known to combine probiotics, prebiotics, and acidified drinking water together to provide synergistic health benefits.